Target cell-specific non-viral vectors for inserting genes into cells, pharmaceutical compositions comprising such vectors and their use

ABSTRACT

Target cell-specific, non-viral vectors for inserting genes into cells, pharmaceuticals composition comprising such vectors, and methods of their use. Target cell-specific, non-viral vectors for inserting at least one gene into cells of an organism, comprising a complex comprising the following components: 
     a) a non-viral carrier for the gene to be inserted, 
     b) a ligand which can bind specifically to the desired target cell, 
     c) a fusion protein for the penetration of the vector into the cytoplasm of the target cell, and 
     d) the gene to be introduced 
     are disclosed. Vectors of this nature are used, for example, in gene therapy.

This application is a continuation of application Ser. No. 08/799,825,filed Feb. 13 1997, now U.S. Pat. No. 5,916,803.

BACKGROUND OF THE INVENTION

The present invention relates to target cell-specific non-viral vectors,to pharmaceutical compositions that comprise such vectors, and to theuse of these vectors in gene therapy.

The aim of gene e therapy is to insert a foreign gene(s) into the cell sof an organism in order either to switch off defective genes, to replacea defective gene with an intact gene, or to enable these cells to form aprotein that possesses a prophylactic or therapeutic effect.

Vectors for insertion of genes into eukaryotic cells, b based on viruses, are well known in the art. Viruses have developed a differentiatedsystem by means of which they bind specifically to cells by means ofcoat proteins, and, after being endocytosed via endosomes, are able topenetrate the membrane of these endosomes and reach the interior of thehost cells. Viruses have therefore been used as carriers for insertingforeign genes into the cell. This technology, in its differentvariations, and the viruses that are used for this purpose, have alreadybeen described in detail (see the reviews of Hodgson, Bio/Technology, 3:222 (1995); and Jolly, Cancer Gene Therapy, 1: 51 (1994)).

The principle underlying this technology is that parts of the viral geneare replaced by the desired foreign gene so that a viral vector isproduced. As a rule, viral vectors are no longer able to replicate, dueto the manipulation. However, all the genes that encode the viral coatproteins and regulate the expression of these viral genes must bepresent to enable these viral vectors to replicate.

It has been found, however, that viral vectors can give rise toproblems, particularly when being used in humans. There is the danger ofrecombination with wild-type viruses of the same species, as a result ofwhich pathogenic viruses might be produced. Furthermore, viral coatproteins can trigger immune reactions in the recipient. As viral vectorstake the same route of infection in the cell as do the correspondingwild viruses, there is the danger of the host genes being mutated as aresult of the foreign genes being integrated into the host chromosomes(activation of quiescent genes, destruction of active genes).

A further disadvantage of viral vectors is that the geometry of theviruses restricts their ability to accommodate many foreign genes.

In view of these limitations and dangers of viral vectors, attempts havebeen made to find virus-independent methods of inserting genes intocells. The principle underlying one of these methods is fusing thenegatively charged cell membrane with the negatively charged gene sothat the gene is taken up by the cell, and penetrates into the cytoplasmthrough the endosomal membrane or the lysosomal membrane. Apart fromdeveloping physical (enclosure of gene particles, osmotic, thermal orelectrical alterations to the cell membrane) or chemical (organicsolvents, detergents, enzymes) methods for altering the cell membrane,gene carriers have been developed that mediate fusion of the genes withthe cell membrane. These carriers include liposomes, cationicpolypeptides, dendrimeric polymers or cationic amphiphilic substances(for reviews, see Behr, Bioconjugate Chem., 5: 382 (1994); Afione etal., Clin. Pharmakokinet., 28: 181 (1995) and Felgner, Adv. DrugDelivery Rev., 5: 163 (1990)).

Synthetic cationic amphiphilic substances, such asdioleoyloxypropyltrimethylammonium bromide (DOTMA) in a mixture withdioleoylphosphatidylethanolamine (DOPE) or lipopolyamine (see Behrabove), have gained considerable importance in this type of charged genetransfer. The mechanism of action of these cationic amphiphilicsubstances or substance mixtures is that, due to an excess of cationiccharge, they both complex with the negatively charged genes and bind tothe anionic cell surface. The amphiphilic character of these carriersleads to fusion with the cell membrane. However, the transfection ratewhich can be achieved is still markedly less than when using viralvectors. Furthermore, the excess cationic charge on the complexescomposed of non-viral carriers and DNA is neutralized, after in-vivoadministration, by anionic biological substances (proteins, heparins,etc.), thereby impairing binding to cells.

It, therefore, is an object of this invention to provide a means forinserting a foreign gene into a eukaryotic cell that avoids thedrawbacks of prior art methods. This and related objects have beenachieved by the invention described below.

SUMMARY OF THE INVENTION

The present invention is based in part on the concept that cells cantake up genes through the process of endocytosis. The endocytosisprocess is normally followed by enzymic degradation of the foreign genesin the endosomes or lysosomes. Only those genes that can evade thisenzymic degradation and can penetrate through the membrane of theendosomes/lysosomes into the cytoplasm and/or into the cell nucleus areable to be expressed by transcription. In the case of the novel targetcell-specific vectors described herein, the local concentration of thevectors at the target cell is increased in vivo as the novel vectors areprovided with target cell-specific ligands.

The present invention relates, therefore, to target cell-specificnon-viral vectors for inserting at least one gene into cells of anorganism, which vectors comprise the following components:

(a) a non-viral carrier for the gene to be inserted,

(b) a ligand which can bind specifically to the desired target cell,

(c) a fusion protein for the penetration of the vector into thecytoplasm of the target cell, and

(d) the gene to be introduced.

In the novel vectors, the individual components of the targetcell-specific vector are bonded to each other covalently and/or by meansof adsorptive bonding. The present invention also relates to apharmaceutical composition comprising the above vector.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The non-viral carrier (a) (see description above) for the gene, that isused in accordance with the invention, is preferably a protein,polypeptide, polysaccharide, phospholipid, cationic lipid, glycoprotein,lipoprotein or lipopolyamine that can be cationized by introducingpositively charged side groups, with the bonding between the non-viralcarrier and a positively charged side chain being effected by adsorptiveor covalent bonding. Furthermore, the carrier can be given amphiphilicproperties by an additional adsorptive or covalent bonding-on oflipophilic side groups. In a particularly preferred embodiment, thenon-viral carrier (a) is albumin or xylan.

The ligand (b) that is employed in accordance with the invention isselected on the basis of specific binding to the outer membrane of aparticular animal or human cell. For binding specifically to endothelialcells, ligand (b) is preferably selected from the group consisting of amonoclonal antibody, or a fragment specific for endothelial cells, aglycoprotein that carries mannose terminally, glycolipid,polysaccharide, cytokine, growth factor, adhesion molecules andglycoproteins from the coats of viruses that possess a tropism forendothelial cells. The last-named is a particularly preferredembodiment.

In another preferred embodiment, ligand (b) that binds specifically tosmooth muscle cells is selected from the group consisting of amonoclonal antibody, or its fragments thereof, that specifically bind toactin, cell membrane receptor, a growth factor, and a glycoprotein fromthe coats of viruses that possesses tropism for smooth muscle cells. Thelast-named is a particularly preferred embodiment.

In a further preferred embodiment, a ligand (b) that binds specificallyto macrophages and/or lymphocytes is selected from the group consistingof a monoclonal antibody that is specific for a membrane antigen onmacrophages or lymphocytes, an intact immunoglobulin or Fc fragments ofpolyclonal or monoclonal antibodies that are specific for membraneantigens on macrophages and lymphocytes, cytokine, growth factor, apeptide carrying mannose terminally, protein, lipid, polysaccharide andglycoprotein from the coat of virus, in particular the HEF protein ofinfluenza C virus having a mutation in nucleotide position 872, or HEFcleavage products of influenza C virus which contain the catalytic triadserine-71, histidine 368 or 369 and aspartic acid 261. The last named isthe particularly preferred embodiment.

Ligand (b) that binds specifically to glial cells is selected from thegroup consisting of an antibody or antibody fragment thereof that bindsspecifically to membrane structures of glial cells, an adhesionmolecule, a peptide carrying mannose terminally, a protein, a lipid or apolysaccharide, growth factor, and a glycoprotein from the coat of avirus that possesses a tropism for glial cells. The last named isparticularly preferred.

In a further preferred embodiment, ligand (b) binds specifically tohematopoietic cells, and is selected from the group consisting of anantibody or antibody fragment that is specific for a stem cell factor,IL-1 (in particular receptor type I or II), IL-3 (in particular receptortype α or β), IL-6 or GM-CSF receptor, an intact immunoglobulin or Fcfragment thereof that exhibits this specificity, growth factor such asSCF, IL-1, IL-3, IL-6 and GM-CSF, and Hepatocyte Growth Factor and afragment thereof that binds to the corresponding receptor.

In yet another preferred embodiment, ligand (b) can bind specifically toleukemia cells and is selected from the group consisting of an antibodyor antibody fragment thereof, an immunoglobulin or Fc fragment that bindspecifically to membrane structures on leukemia cells such as CD13,CD14, CD15, CD33, CAMAL, sialosyl-Le, CD5, CD1e, CD23, M38, IL-2receptors, T-cell receptors, CALLA or CD19, and also growth factors, orfragments that derive from them, and retinoids.

A ligand (b) that can bind specifically to virus-infected cells ispreferably selected from the group consisting of an antibody or antibodyfragment thereof, and an intact immunoglobulin or Fc fragment that isspecific for a viral antigen which, after infection with the virus, isexpressed on the cell membrane of the infected cell.

Finally, ligand (b) can also be a ligand that binds specifically tobronchial epithelial cells, and sinusoidal cells of the liver orhepatocytes, and is selected from the group consisting of transferring,an asialoglycoprotein such as asialoorosomucoid, a neoglycoprotein,galactose, insulin, a peptide carrying mannose terminally, protein,lipid, polysaccharide, intact immunoglobulin or Fc fragment which bindspecifically to the target cells, and glycoproteins from the coats ofviruses that bind specifically to the target cells. The last-namedembodiment is particularly preferred.

A fusion protein (c) is selected from the group consisting ofhemagglutinin of influenza A or A viruses, the HA2 component of thehemagglutinin of influenza A or B viruses, a peptide analogue thereof,the M2 protein of influenza A viruses, the HEF protein of influenza Cviruses, a transmembrane protein of filoviruses, a transmembraneglycoprotein of rabies virus, vesicular stomatitis virus, Semliki ForestVirus, tickborn encephalitis virus, a fusion protein of HIV virus,Sendai virus, respiratory syncytial virus, and a fragment of said viralfusion proteins or of transmembrane glycoprotein from which thetransmembrane region was removed.

In a preferred embodiment, a fusion protein (c) is selected from thegroup consisting of hemagglutinin or influenza A or B viruses, the HA2component of the hemagglutinin or influenza A or B viruses and peptideanalogs thereof, the M2 protein of influenza A viruses, the HEF proteinof influenza C viruses, a transmembrane protein of filoviruses such asMarburg virus or Ebola virus, a transmembrane glycoprotein of rabiesvirus, vesicular stomatitis virus, Semliki Forest virus, tickbornencephalitis virus, a fusion protein of HIV virus, Sendai virus (inparticular the F1 component) or respiratory syncytial virus (inparticular the gp37 component) and fragments of these viral fusionproteins or of the transmembrane glycoproteins that contain thefusiogenic peptides.

In a preferred embodiment, the gene (d) which is to be introduced is inthe form of a plasmid.

The novel vectors can be employed as a pharmaceutical or a constituentof a pharmaceutical, with the vectors preferably being used forpreparing a pharmaceutical for the intravenous, intraarterial,intraportal, intracranial, intrapleural, intraperitoneal or localintroduction of a desired gene into specific target cells.Pharmaceutically acceptable carriers for the novel vectors are thoselisted in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 1988, hereby incorporated by reference.

Use of the novel target cell-specific vectors ensures that, after thevector has been administered parenterally, preferably intravenously orintraarterially, its concentration at the target cell can be raised andthe rate at which the target cell is transfected can consequently beincreased. This advantage in accordance with the invention is achievedby a synergistic effect of the individual, mutually linked components ofthe novel vectors.

The target cell-specific ligand (b) should exhibit a high specificityfor the desired target cells. In a preferred embodiment, ligand is aviral coat protein which is specific for particular cells. A suitableviral coat protein is selected on the basis of the desired target cell.As these target cell-specific ligands, in particular the viral coatproteins, are coupled to the carrier for the desired gene, the desiredgene binds to the target cell by way of the target cell-specific ligandand becomes enriched at the target cell.

The non-viral carriers (a) for the gene are preferably those compoundsthat are known to have a long half-life in the blood. As a result ofthis relatively long half-life in the blood, the target cell is exposedfor as long as possible to as high a concentration of the vector aspossible in order thereby to achieve the maximum possible binding ofvectors to the target cells by means of the target cell-specificligands. In a particularly preferred embodiment, these non-viralcarriers (a) are cationized to enable them to complex with thenegatively charged DNA.

In addition, the novel vectors may contain a fusion protein that isresponsible for penetration of the vector out of the endosomes orlysosomes and into the cytoplasm of the host cell. Within the context ofthe present invention, fusion proteins are defined as those proteinsthat enable the vector to enter the cytoplasm of the target cell. Fusionproteins of this nature are known, especially from viral sources.

The gene (d) that is to be introduced by the novel vectors can be in theform of a nucleic acid containing the corresponding gene which, ifnecessary, is provided with the appropriate regulatory regions such aspromoters, etc. In a preferred embodiment, the gene that is to beintroduced is in the form of a plasmid.

The non-viral carriers for the gene, that can be employed in accordancewith the invention, are known in the art (for reviews, see Cotten etal., Curr. Biol., 4: 705 (1993); Behr, Acc. Chem. Res., 26: 274 (1993) ;Felgner, Adv. Deliv. Rev., 5: 163 (1990); Behr, Bioconjugate Chem., 5:382 (1994); Ledley, Hum. Gene Ther. 6: 1129 (1995) all of which arehereby incorporated by reference). Those that are preferably employedwithin the context of the present invention are liposomes, cationicliposomes that are prepared using cationic lipids such as stearyl aminesin a mixture with neutral phospholipids, dioctadecyldimethylammoniumbromide (DDA) in a mixture with neutral phospholipids,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium bromide (DOTMA),3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol),1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),dimethyldioctadecylammonium bromide (DDAB) and1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP).

Cationic polypeptides and proteins, such as polylysine, protaminesulfates, histones, polyornithine and polyarginine, are also suitable asnon-viral carriers, as are cationic amphiphilic lipopolyamines such asdioctadecylamidoglycylspermine (DOGS),dipalmitoylphosphatidylethanolamidospermine (DPPES),N-t-butyl-N′-tetradecyl-3-tretradecylaminopropionamide (diC14-amidine),DoTB, ChoTB, DoSC, ChoSC, LPLL, DEBDA, DTAB, TTAB, CTAB or TMAG,orcationic polysaccharides such as diethylaminoethyldextran, and alsocationic organic polymers such as Polybrene.

In a further preferred embodiment, formulations of cationic lipids andlipopolyamines (complexed with DNA) can, for the purpose of increasingthe transfection rate, be supplemented by admixing neutral phospholipidssuch as dioleoylphosphatidylethanolamine (DOPE).

In a particularly preferred embodiment, the non-viral carriers arecompounds whose parent substances are cationic or cationizedwater-soluble polypeptides, proteins, glycoproteins, lipoproteins orpolysaccharides that exhibit amphiphilic behavior due to theintroduction of (where appropriate additional) lipophilic groups. Theparent substances are preferably water-soluble carriers, such asproteins, glycoproteins, lipoproteins or polysaccharides. In aparticularly preferred embodiment, the carrier is albumin or xylan.

Structural units that have a positive charge and that can be bound tothe parent substance are suitable for use as cationic groups.Preferably, the cationic groups are structural units that exhibit amino,guanidino or imidazolyl functions under physiological conditions. Thecationic groups can be coupled to the parent substances using well-knownmethods of conjugation. For example, coupling with diamines, such asethylenediamine or hexamethylenediamine, is suitable for aminofunctions. Free amino groups can also be methylated with methyl iodide,or unilaterally reacted glutaraldehyde groups can be reacted with GirardT reagent [see, e.g., Roser, Dissertation, University of Basle (1990)hereby incorporated by reference].

Guanidino and imidazolyl groups may be introduced by coupling to thecorresponding basic amino acids using the methods that are describedbelow. Preference is given to coupling to albumin using glutaraldehyde(Roser, above).

The number of cationic groups that have to be introduced depends on themagnitude of the anionic charge of the gene or the nucleotide sequencewith which the carrier is to be complexed. Preferably, the complex as awhole should have a neutral or cationic charge.

All structural units that lead to an increase in solubility in organicsolvents, for example octanol, are regarded as lipophilic groups.Unsaturated fatty acids, for example oleic acid, that are used asesters, acid chlorides and acid anhydrides are, in particular, regardedas lipophilic groups. Lipophilic groups are introduced using well-knownmethods of conjugation, for example by acylation, i.e. the reaction ofacid chorides, acid anhydrides and esters with primary and secondaryamines, as described in Seebach, Angew., Chemie, 81: 690 (1969) andSatchell, Quart. Rev., 17: 160 (1963) hereby incorporated by reference.The number of lipophilic groups that have to be introduced depends onthe degree of lipophilicity of the parent substance.

A large number of molecules can be used as tissue-targeting ligand (b).As described above, the choice of suitable ligand depends on the targetcells for which the vector is to be specific. As a rule, the ligands areproteins, polypeptides or glycoproteins that exhibit a high specificaffinity for membrane constituents on selected cells (target cell).Depending on the target cell, ligands that can be used within thecontext of the present invention include:

1) Ligands for Endothelial Cells

a) Non-viral Ligands (b)

Substances that preferably bind to the surface of endothelial cells, inparticular proliferating endothelial cells, are used as ligands. Thesesubstances include antibodies or antibody fragments that are directedagainst membrane structures of endothelial cells, as have beendescribed, for example, by Burrows et al. (Pharmac. Ther., 64: 155(1994)); Hughes et al. (Cancer Res., 49: 6214 (1989)) and Maruyama etal., (PNAS-USA, 87: 5744 (1990) all of which are hereby incorporated byreference. In particular, these substances include antibodies againstthe VEGF receptors.

Murine monoclonal antibodies should preferably be employed in humanizedform. Humanization is effected in the manner described by Winter et al.(Nature, 349: 293 (1991)) and Hoogenbooms et al. (Rev. Tr. Transfus.Hemobiol., 36: 19 (1993) both of which are hereby incorporated byreference). Antibody fragments are prepared by routine methods, forexample in the manner described by Winter et al. above; Hoggenbooms etal. above; Girol, Mol. Immunol., 28: 1379 (1991) or Huston et al.,Intern. Rev. Immunol., 10: 195 (1993) all of which are herebyincorporated by reference.

In addition, the ligands include all active compounds which bind tomembrane structures or membrane receptors on endothelial cells. Forexample, these active compounds include kinins, and analogs and homologsof kinins, that bind to kinin receptors, and also substances thatcontain mannose terminally, and also IL-1 or growth factors, or theirfragments or constituent sequences thereof, which bind to receptorswhich are expressed by endothelial cells, such as HGF, PDGF, bFGF, VEGFor TGFβ (Pusztain et al., J. Pathol., 169: 191 (1993) herebyincorporated by reference). Furthermore, these active compounds includeadhesion molecules which bind to activated and/or poliferatingendothelial cells. Adhesion molecules of this nature, for example Slex,LFA-1, MAC-1, LECAM-1 or VLA-4, have already been described (reviews inAugustin-Voss et al., J. Cell. Biol., 119: 483 (1992); Pauli et al.,Cancer Metast. Rev., 9: 175 (1990); Honn et al., Cancer Metast. Rev.,11: 353 (1992) hereby incorporated by reference).

b) Viral-derived Ligands

The ligands within the meaning of this invention also includeglycoproteins from the coats of viruses that possess a tropism forendothelial cells. Examples of these viruses are:

filoviruses, for example

Marburg virus

with its coat protein GP (glycoprotein) and sGP (second glycoprotein)

(Kiley et. al., J. General Virology, 69: 1957 (1988); Will et al., J.Virol., 67: 1203 (1993); Schnittler et al., J. Clin. Invest., 91: 1301(1993); Feldmann et al., Virus Res. 24: 1 (1992) all of which are herebyincorporated by reference)

or Ebola virus

in each case with its coat protein GP and sGP

(Schnittler et al., J. Clin. Invest., 91: 1301 (1993); Volchov et al.,Virol., 214: 421 (1995); Jahrling et al., Lancet, 335: 502 (1990);Feldmann et al., Arch. Virol., 7: 81 (1993); Geisbert et al., J. Comp.Path., 106: 137 (1992) all of which are hereby incorporated byreference)

cytomegalovirus,

particularly with its gB protein

(Waldman et al., Transplant. Proc., 27: 1269 (1995); Sedmak et al.,Transplant, 58: 1379 (1994); Sedmak et al., Archives Virol, 140: 111(1995); Koskines, Transplant, 56: 1103 (1993); Scholz et al., Hum.Immunol. 35: 230 (1992); Alcami et al., J. Gen. Virol., 72: 2765 (1991);Poland et al., J. Infect. Dis., 162: 1252 (1990); Ho et al., J. Infect.Dis., 150: 956 (1984); Spaete et al., J. Virol., 64: 2922 (1990) all ofwhich are hereby incorporated by reference)

herpes simplex virus type I

(Etingin et al., PNAS 90, 5153 (1993); Key et al., Lab. Invest., 68: 645(1993); Kubota et al., J. Immunol., 138: 1137 (1987) all of which arehereby incorporated by reference)

the HIV-1 virus

(Scheylovitova et al., Arch. Virol., 140: 951 (1995); Lafon et al.,AIDS, 8: 747 (1994); Re et al., Microbiologica, 14: 149 (1991) all ofwhich are hereby incorporated by reference)

measles virus

(Mazure et al., J. Gen. Virol., 75: 2863 (1994) hereby incorporated byreference)

Hantaan virus

(Pensiero et al., J. Virol., 66: 5929 (1992); Zhu, Chinese Med. J., 68:524 (1988) both of which are hereby incorporated by reference)

alphaviruses, such as Semliki Forest virus

(Jakob, J. Med. Microbiol., 39: 26 (1993) hereby incorporated byreference)

epidemic hemorrhagic fever virus

(Yi, Chinese J. Pathol., 21: 177 (1992) hereby incorporated byreference)

poliovirus

(Condere et al., Virol., 174: 95 (1990) hereby incorporated byreference)

enteroviruses (such as Echo 9, Echo 12, Coxsackie B3)

(Kirkpatrick et al., Am. J. Pathol., 118: 15 (1985) hereby incorporatedby reference).

2) Ligands for Smooth Muscle Cells

a) Non-viral Ligands (b)

Examples of non-viral ligands (b) that may be used are antibodies orantibody fragments which are directed against membrane structures ofsmooth muscle cells. These include:

the antibody 10F3 (Printseva et al., Exp. Cell Res., 169: 85 (1987);American J. Path. 134, 305 (1989) both of which are incorporated hereinby reference)

Antibodies against actin

Antibodies against angiotensin II receptors

Antibodies against receptors for growth factors or antibodies directed,for example, against

EGF receptors

PDGF receptors

FGF receptors

against endothelin A receptors.

In addition, these ligands include all active substances that bind tomembrane structures or membrane receptors on smooth muscle cells(reviews in Pusztai et al., J. Pathol., 169: 191 (1993), Harris, CurrentOpin. Biotechnol., 2: 260 (1991) all of which are hereby incorporated byreference). For example, these active substances include growth factorsor their fragments, or constituent sequences thereof, which bind toreceptors that are expressed by smooth muscle cells, for example

PDGF

EGF

TGFβ

TGFa

FGF

endothelin A

b) Viral-derived Ligands (b)

However, ligands within the meaning of this invention include, inparticular, glycoproteins from the coats of those viruses which possessa tropism for smooth muscle cells. An example of these viruses iscytomegalovirus (Speir et al., Science 265, 391: (1994) herebyincorporated by reference).

3) Choice of the Ligand for Macrophages and Lymphocytes

a) Non-viral Ligands (b)

The ligands that bind specifically to the surface of macrophages andlymphocytes include antibodies or antibody fragments that are directedagainst membrane structures of immune cells, as described, for example,by Powelson et al., Biotech. Adv., 11: 725 (1993) hereby incorporated byreference.

In addition, the ligands also include monoclonal or polyclonalantibodies or antibody fragments which bind, by their constant domains,to Fc-g receptors or FC-e receptors of immune cells (Rojanasakul et al.,Pharm. Res., 11: 1731 (1994) hereby incorporated by reference); theseinclude, in particular, the Fc fragment of human polyclonalimmunoglobulin. Fc fragments of this nature are prepared, for example,in accordance with the methods of Haupt et al., Klin. Wschr., 47: 270(1969); Kranz et al., Dev. Biol. Standard, 44: 19 (1979); Fehr et al.,Adv. Clin. Pharmac., 6: 64 (1974); and Menninger et al., Immunochem. 13:633 (1976) all of which are hereby incorporated by reference.

These ligands furthermore include all substances that bind to membranestructures or membrane receptors on the surface of immune cells.Examples of these substances are the cytokines IL-1, IL-2, IL-3, IL-4,IL-6, IL-10, TNFa, GM-CSF and M-CSF, and also growth factors such asEGF, TGF, FGF, IGF or PDGF, HGF, or their fragments or constituentsequences thereof, which bind to receptors which are expressed by cellsof this nature.

These ligands also include ligands that bind to cell membrane structuressuch as the mannose 6-phosphate receptor on macrophages in spleen,liver, lung and other tissues. These ligands and membrane structureshave been reviewed by Perales et al., Eur. J. Biochem., 226: 255 (1994)hereby incorporated by reference.

b) Viral Ligands

However, the ligands within the meaning of this section include, inparticular, glycoproteins from the coats of those viruses which possessa tropism for lymphocytes and/or macrophages.

Examples of viruses that infect macrophages include:

HIV-1,

particularly those strains having mutations in the V3 region of gp120which lead to increased binding to macrophages, for example as describedby Kim et al., J. Virol., 69: 1755 (1995); Valentin et al., J. Virol.,68: 6684 (1994); Collin et al., J. Gen. Virol., 75: 1597 (1994); Shoidaet al., PNAS, 89: 9434 (1992); Chesebro et al. , J. Virol., 66: 6547,(1992); Shaw et al., J. Virol., 66: 2577 (1992); Liu et al., J. Virol.,64: 6148 (1990); Broder et al., PNAS, 92: 9004 (1995); Cangue et al.,Virol., 208:, 779 (1995), all of which are hereby incorporated byreference.

HIV-2

(Valentin et al., J. Virol., 68: 6684 (1994) hereby incorporated byreference)

hantaviruses, for example the Punmala virus

(Temonen et al., Virol., 206: 8 (1995), hereby incorporated by reference

cytomegalovirus

(Fajac et al., Am. J. Resp. Crit. Care Med., 149: 495 (1994); Kondo etal., PNAS, 91: 11879 (1994); Ibanez et al., J. Virol., 65: 6581 (1991)all of which are hereby incorporated by reference)

respiratory syncytial virus

(Becker et al., Am. J. Resp. Cell Mol. Biol., 6: 369 (1992); Roberts,Infect. Immun., 35: 1142 (1982) both of which are hereby incorporated byreference)

herpes simplex virus

(Plaeger-Marshall et al., Pediatric Res., 26: 135 (1989) herebyincorporated by reference)

filoviruses

(Schnittler et al., J. Clin. Invest., 91: 1301 (1993); Zaki, Eur. Conf.Tropical Med., p2 (A22), Hamburg, Germany (1995) both of which arehereby incorporated by reference.

Examples of viruses that infect lymphocytes include:

varicella zoster virus (VZV)

VZV infects T cells in particular

(Moffat et al., J. Virol., 69: 5236 (1995) hereby incorporated byreference)

herpesvirus 6 (HHV-6).

HHV-6 infects T cells in particular

(Takahashi et al., J. Virol., 63: 3161 (1989) hereby incorporated byreference; Lusso et al., J. Exp. Med., 181: 1303 (1995); Frenkel et al.,Adv. Exp. Med. Biol., 278: 1 (1990) hereby incorporated by reference)

rabies virus

Rabies virus coat protein binds, in particular, to TH2 cells(Martinez-Arends et al., Clin. Immunol. Immunopath., 77: 177 (1995)hereby incorporated by reference)

HIV-1

The gp 120 glycoprotein binds preferentially to the CD4 molecule of Tcells

(Heinkelein et al., J. Virol., 69: 6925 (1995) hereby incorporated byreference)

HTLV-II

HTLV-II infects B cells in particular

(Casoli et al., Virol., 206: 1126 (1995) hereby incorporated byreference)

HTLV-I

HTLV-I infects T cells in particular

(Persaud et al., J. Virol., 69: 6297 (1995); Boyer et al., CellImmunol., 129: 341 (1990) both of which are hereby incorporated byreference)

influenza C viruses

Influenza C viruses bind, by way of the hemagglutininesterase fusion(HEF) protein, to N-acetyl-9-β-acetylneuraminic acid (Neu 5,9 Ac), whichoccurs preferentially on B lymphocytes and less, or not at all, on Tlymphocytes

(Herrler et al., EMBO-J., 4: 1503 (1985); Kamerling et al., BBA, 714:351 (1982); Rogers et al., J. Biol. Chem., 261: 5947 (1986) herebyincorporated by reference)

influenza C viruses having a mutation in nucleotide position 872

(which encodes position 284 of the amino acid sequence of the HEF), forexample a replacement of the threonine by isoleucine.

The surface protein HEF having this mutation has a markedly greateraffinity for the N-acetyl-9-0-acetylneuraminic acid receptor than doesthe wild-type virus.

(Szepanski et al., Virol., 188: 85 (1992) hereby incorporated byreference)

HEF cleavage products of influenza C virus which contain the structurefor binding to N-acetyl-9-β-acetylneuraminic acid.

This binding structure is defined by the catalytic triad serine 71,histidine 368 or 369 and aspartic acid 261

(Pleschka et al., J. Gen. Virol., 76: 2529 (1995) hereby incorporated byreference)

Epstein-Barr virus.

EBV infects B cells in particular

(Miller-Yale, J. Biol. Med., 55: 305 (1982); Garzelli et al., Immunol.Lett., 39: 277 (1994); Counter et al., J. Virol., 68: 3410 (1994); Wanget al., J. Virol., 62: 4173 (1988) all of which are incorporated hereinby reference)

herpes simplex virus 2.

HSV-2 infects T cells in particular (Kucera et al., Viral Immun., 2: 11(1989) hereby incorporated by reference)

measles virus

(Jacobson et al., J. Gen. Virol., 63: 351 (1982) hereby incorporated byreference)

4) Choice of the Ligands for Glial Cells

a) Non-viral Ligands (b)

Substances that bind to the surface of glial cells are also to beregarded as ligands. These substances include antibodies or antibodyfragments that are directed against membrane structures of glial cells,as reported, for example, by Mirsky et al., (Cell and Tissue Res., 240:723 (1985)); by Coakham et al. , (Prog. Exp. Tumor Res., 29: 57 (1985))and by McKeever et al. (Neurobiol., 6: 119 (1991) all of which arehereby incorporated by reference). These membrane structures furthermoreinclude neural adhesion molecules such as N-CAM, in particular itspolypeptide chain C (Nybroe et al., J. Cell Biol., 101: 2310 (1985)hereby incorporated by reference).

These ligands furthermore include all active compounds that bind tomembrane structures or membrane receptors on glial cells. Examples ofthese active compounds are substances which carry mannose terminally andbind to the mannose 6-phosphate receptor (Perales et al., Eur. J.Biochem., 226: 225 (1994), insulin and insulin-like growth factor(Merrill et al., J. Clin. Endocrin. Metab., 71: 199 (1990)), PDGF (Ek etal., Nature, 295: 419 (1982), hereby incorporated by reference, andthose fragments of these growth factors which bind to the affiliatedmembrane receptors.

b) Viral-derived Ligands (b)

The ligands within the meaning of the invention include, in particular,glycoproteins from the coats of those viruses that possess a tropism forglial cells.

Examples of these viruses are:

HIV-1 subtype JRF1

(Sharpless et al., J. Virol. 66, 2588 (1992), hereby incorporated byreference

herpes simplex virus I

(Xie et al., Eye Science (Yen Ko Hsueh Pao) 10, 67 (1994); Genis et al.,J. Exp. Med. 176, 1703 (1992) both of which are hereby incorporated byreference)

5) Non-viral Ligands for Hematopoietic Cells

The ligands include antibodies or antibody fragments which are directedagainst receptors which are expressed, e.g., on blood cells, that areonly slightly differentiated.

Antibodies of this nature have been described for the followingreceptors include:

stem cell factor receptor

IL-1 receptor (Type I)

IL-1 receptor (Type II)

IL-3 receptor a

IL-3 receptor β

IL-6 receptor

GM-CSF receptor.

In addition, these ligands also include monoclonal or polyclonalantibodies or antibody fragments that bind, by their constant domains,to FC-g receptors of immune cells (Rojanasakul et al., Pharm. Res. 11,1731 (1994)).

The ligands also include substances that bind to membrane structures ormembrane receptors on the surface of blood cells which are only slightlydifferentiated. Examples of these substances are growth factors such asSCF, IL-1, IL-3, IL-6 or GM-CSF, or their fragments or constituentsequences thereof, which bind to receptors which are expressed by cellsof this nature.

6) Non-viral Ligands for Leukemia Cells and Tumor Cells

The ligands that bind to the surface of leukemia cells includeantibodies or antibody fragments which are directed against membranestructures of leukemia cells. A large number of such monoclonalantibodies have already been described for diagnostic and therapeuticmethods (Reviews in Kristensen, Danish Medical Bulletin, 41: 52 (1994);Schranz, Therapia Hungarica, 38: 3 (1990); Drexler et al., Leuk. Rex.,10: 279 (1986); Naeim, Dis. Markers, 7: 1 (1989); Stickney et al.,Current Op. Oncol., 4: 847 (1992); Drexler et al., Blut, 57: 327 (1988);Freedment et al., Cancer Invest., 9: 69 (1991) all of which areincorporated herein by reference). The following monoclonal antibodies,or their antigen-binding antibody fragments, are, for example, suitablefor use as ligands, depending on the type of leukemia:

Monoclonal Antibodies Membrane described Cells antigen by AML CD13Kaneko et al., Leuk. Lymph., 14: 219 (1994) — Muroi et al., Blood, 79:713 (1992) CD14 Ball, Bone Marrow Transplnt., 3: 387 (1988) CD15 Guyotatet al., Bone Marrow Transplant., 6: 385 (1990) CD33 Jurcic et al.,Leukemia, 9: 244 (1995) Caron et al., Cancer, 73: 1049 (1994) CAMALShellard et al., Exp. Hematol., 19: 136 (1991) Sialosyl-Le Muroi et al.,Blood, 79: 713 (1992) All of the references are hereby incorporated byreference B-CELL CD5 Kaminski et al., Cancer Treat. Res., 38: 253 (1988)Tassone et al., Immunology Lett., 39: 137 (1994) CD1c Orazi et al., Eur.J. CD23 Hematol., 47: 28 (1991) All of the above references are herebyincorporated by reference Idiotypes and isotypes of the membraneimmunoglobulins Schroeder et al., Immunol. Today, 15: 289 (1994), herebyincorporated by reference. T-CELL CD33 Imai et al., J. Immunol., 151:6470 (1993) IL-2 Waldmann et al., Blood, receptors 82: 1701 T cell(1993) receptors All of the above references are hereby incorporated byreference ALL CALLA Morishima et al., Bone Marrow Transplant., 11: 255(1993) CD19 Anderson et al., Blood, Non-Hodgkin 80: 84 (1993) LymphomaOkazaki et al., Blood, 81: 84 (1993)

All of the above references are hereby incorporated by reference.

The non-viral ligands for tumor cells include antibodies, and fragmentsof these antibodies, which are directed against membrane structures ontumor cells. Antibodies of this nature have been reviewed, for example,by Sedlacek et al., Contrib. to Oncol., 32: Karger Verlag, Munich (1988)and Contrib. to Oncol., 43: Karger Verlag, Munich (1992) both of whichare hereby incorporated by reference.

Additional examples are antibodies against:

sialyl Lewis

(Ohta et al., Immunol. Lett., 44: 35 (1995) hereby incorporated byreference)

peptides on tumors which are recognized by T cells

(Maeurer et al., Melanoma Res., 6: 11 (1996); Coulie, Stem Cells, 13:393 (1995); Stoh et al., J. Biochem, 119: 385 (1996); Slingluff et al.,Curr. Opin. Imunol., 6: 733 (1994) all of which are hereby incorporatedby reference)

proteins which are expressed from oncogenes

(Cheever et al., Immunol. Rev., 145: 33 (1995); Talarico et al., PNAS,87: 4222 (1990))

gangliosides such as GD3, GD2, GM2, 9-0-acetyl GD3 and fucosyl GM1

(Helling et al., Cancer Res., 55: 2783 (1995); Livingston et al.,Vaccine, 11: 1199 (1993); Vaccine, 12: 1275 (1994); Livingston et al.,Cancer Immunol. Immunother., 29: 179 (1989); Gnewuch et al., Int. J.Cancer, 8: 125 (1994); Jennemann et al., J. Biochem., 115: 1047 (1994);Ravindranath et al., Cancer Res., 49: 3891 (1989) all of which arehereby incorporated by reference)

blood group antigens and their precursors

(Springer et al., Cancer, 37: 169 (1976); Carbohydrate Res., 179: 271(1988); Molec. Immunol., 26: 1 (1989); Fung et al., Cancer Res., 50:4308 (1990) all of which are hereby incorporated by reference)

antigens on polymorphic epithelial mucin

(PEM; Burchell et al., Cancer Surreys, 18: 135 (1993) herebyincorporated by reference)

antigens on heat shock proteins

(Blackere et al., J. Immunother., 14: 352 (1993) hereby incorporated byreference).

The murine monoclonal antibodies are preferably to be employed inhumanized form. The humanization is effected as already described. Asalready described, antibody fragments are prepared in accordance withthe state of the art.

The ligands additionally include all active compounds which bind tomembrane structures or membrane receptors of leukemia cells or tumorcells. Examples of these active compounds are steroid hormones orpeptide hormones, or else growth factors, or their fragments orconstituent sequences thereof, which bind to receptors which areexpressed by leukemia cells or tumor cells.

Growth factors of this nature have already been described (Reviews inCross et al., Cell, 64: 271 (1991); Aulitzky et al., Drugs, 48: 667(1994); Moore, Clin. Cancer Res., 1: 3 (1995); Van Kooten et al., Leuk.Lymph. 12: 27 (1993) all of which are hereby incorporated by reference).For example, they include:

IFNa in non-Hodgkin lymphomas

IL-2, particularly in T cell leukemias

FGF in T cell monocytic, myeloid, erythroid and megakaryoblasticleukemias

TGFβ in leukemias

retinoids, e.g. “retinoic acid” in acute promyelocytic leukemia.

7) Non-viral Ligands (b) for Infected Cells

Ligands for the therapy of infectious diseases include antibodies orantibody fragments that are directed against the agents causing theinfection. For example, in the case of viral infections, these are theviral antigens which are expressed on the cell membrane ofvirus-infected cells.

Antibodies of this nature have been described, for example, for cellsthat are infected with the following viruses:

HBV

HCV

HSV

HPV

HIV

EBV

HTLV.

In addition, the ligands also include monoclonal or polyclonalantibodies, or antibody fragments, which bind, by their constantdomains, to Fc-g receptors or FC-e receptors of immune cells.

The murine monoclonal antibodies are preferably to be employed inhumanized form. The humanization is effected as already described.

The ligands furthermore include all substances which bind to membranestructures or membrane receptors on the surface of virus-infected cells.Examples of these substances include growth factors such as cytokines,EGF, TGF, FGF, HGF or PDGF, or their fragments or constituent sequencesthereof, which bind to receptors which are expressed by cells of thisnature.

8) Ligands for Other Parenchymal Cells

a) Non-viral Ligands (b)

These include ligands that bind to cell membrane structures that areselective for particular tissues. Examples of these ligands are:

Membrane structure Ligands Tissue cells asialoglycoproteinasialoorosomucoid liver cells receptor neoglycoprotein galactosetransferrin transferrin liver, cells of receptor other tissues insulinreceptor insulin macrophages in spleen, liver, lung and other tissuesFc-γ receptors immunoglobulin G reticuloendothelial system and othertissues

These ligands and membrane structures have been reviewed by Perales etal., Eur. J. Biochem., 226: 255 (1994) hereby incorporated by reference.

b) Viral-derived Ligands (b)

However, within the meaning of the invention, these ligands include, inparticular, glycoproteins from the coats of viruses which possess atropism for selected cells, for example for

bronchial epithelial cells

respiratory syncytial virus

(Becker et al., Am. J. Resp. Cell Mol. Biol., 6: 369 (1992) herebyincorporated by reference)

liver cells

hepatitis C virus

(Uchida et al., Pathol. Internat., 44: 832 (1994); Carloni et al., Arch.Virol., 8: 31 (1993); Prince et al., Curr. Stud. Hemat. Blood Trans.,61: 195 (1994) all of which are hereby incorporated by reference)

filoviruses

liver cells bind the Marburg virus, for example, by way of theasialoglycoprotein receptor,

(Becker et al., J. Gen. Virol., 76: 393 (1995) hereby incorporated byreference),

hepatitis B virus

liver cells bind, preferentially by way of the asialoglycoproteinreceptor, to the preS2 and preS1 domain of HBV (Shimizu et al., J. Med.Virol., 20: 313 (1986); Treichel et al., J. Gen. Virol., 75: 3021(1994); Gerlich et al., J. Hepat., 17/3: 10 (1993); Gripon et al.,Virol., 192: 534 (1993); Pontisso et al., Hepatol., 14: 405 (1991);Ochiya et al., PNAS, 86: 1875 (1989) all of which are herebyincorporated by reference)

hepatitis D virus

(Colombo et al., J. Hepatol., 12: 64 (1991) hereby incorporated byreference)

liver sinusoidal cells

hepatitis V virus

HBV is bound by way of fibronectin

(Budhowska et al., J. Virol., 69: 840 (1995) hereby incorporated byreference).

9) Ligands for the Prophylaxis of Infectious Diseases

For the prophylaxis of infectious diseases, all substances which bind tocell membrane structures of macrophages and/or lymphocytes are suitablefor use as ligands. Ligands of this nature have already been described.

Preparation of Viral-derived Ligands (b)

Viral-derived ligands are isolated either by dissolving the envelopeproteins from an enriched viral preparation with the aid of detergents(such as β-D-octylglucopyranoside) and separating them by centrifugation(review in Mannino et al., Bio-Techniques, 6: 682 (1988) herebyincorporated by reference) or else using molecular biological methodswhich are known to the skilled person, as have been described in theliterature cited under II/2/a-h), for example by Pleschka et al. (J.Gen. Virol., 76: 2529 (1995)) and by Szepanski et al. (Virol., 188: 85(1992) hereby incorporated by reference) for the HEF glycoprotein.

Fusion Proteins (c)

Within the context of the present invention, proteins are used thatpossess fusiogenic properties. Proteins of this nature are able to fusedirectly with cell membranes. A number of viruses, for exampleparamyxoviruses, retroviruses and herpesviruses, possess fusiogenic coatproteins (Gaudin et al., J. Gen. Virol., 76: 1541 (1995) herebyincorporated by reference).

Within the meaning of this invention, fusiogenic proteins also includethose glycoproteins that fuse with the cell membrane or endosomes onlyafter having been internalized in endosomes and only at an acid pH.

A number of viruses possess glycoproteins that are responsible both forthe adhesion of the virus and also, subsequently, for the cell membranefusion (Gaudin et al., J. Gen. Virol., 76: 1541 (1995) all of which arehereby incorporated by reference).

Proteins of this nature are formed, for example, by alphaviruses,rhabdo- viruses and orthomyxoviruses.

Viral-derived Fusion Proteins (c)

Viral fusion proteins within the meaning of the invention have beenreviewed by Hughson, Current Biol., 5: 265 (1995); Hoekstra, J.Bioenergetics Biomembranes, 22: 675 (1990); and White, Ann. Rev.Physiol., 52: 675 (1990) all of which are hereby incorporated byreference).

Examples of fusion proteins within the meaning of this invention are:

the hemagglutinin of influenza A or B viruses, in particular the HA2component

(Stegmann et al., J. Biol. Chem., 266: 18404 (1991); Klenk et al.,Virol., 68: 426 (1975); Lazarowitz et al., Viral., 68: 440 (1975);Skehel et al., PNAS, 79: 968 (1982); Bosch et al., Virol., 113: 725(1981) all of which are hereby incorporated by reference)

the M2 protein of influenza A viruses

(Sugrue et al., Virol., 180: 617 (1991); Lamb et al., Cell, 40: 627(1985); Pinto et al, Cell, 69: 517 (1992); Zebedee et al., J. Virol.,56: 502 (1985); Black et al., J. Gen. Virol., 74: 1673 (1993); Whartonet al., J. Gen. Virol., 75: 945 (1994) all of which are herebyincorporated by reference) either alone or employed in combination(Ohuchi et al., J. Virol., 68: 920 (194) which is hereby incorporated byreference) with the hemagglutinin of influenza or with mutants ofneuraminidase of influenza A which lack enzyme activity but which bringabout hemagglutination. (Hausmann et al., J. Gen. Virol., 76: 1719(1995), hereby incorporated by reference)

peptide analogs of the influenza virus hemagglutinin

(Wharton et al., J. Gen. Virol., 69: 1847 (1988) hereby incorporated byreference)

the HEF protein of influenza C virus

The fusion activity of the HEF protein is activated by cleaving the HEFointo the HEF1 and HEF2 subunits

(Herrler et al., J. Gen. Virol., 69: 839 (1988); Kitane et al., Arch.Virol., 73: 357 (1982); Ohuchi et al., J. Virol., 42: 1076 (1982) herebyincorporated by reference)

the transmembrane glycoprotein of filoviruses, such as

Marburg virus

(Feldmann et al., Viral., 182: 353 (1991); Will et al., J. Virol., 67:1203 (1993); Kiley et al., J. Gen. Virol., 69: 1957 (1988); Geyer etal., Glycobiol., 2: 299 (1992) hereby incorporated by reference)

Ebola virus

(Elliott et al., Virol., 147: 169 (1985); Cox et al., J. Infect. Dis.,147: 272 (1983); Kiley et al., J. Gen. Virol., 69: 1957 (1988); Feldmannet al., Arch. Virol., 7: 81 (1993); Volchkov et al., Virol., 214: 421(1995) all of which are hereby incorporated by reference)

the transmembrane glycoprotein of rabies virus

(Whitt et al., Virol., 185: 681 (1991); Gaudin et al., J. Virol., 65:4853 (1991); 67: 1365 (1993); Virology, 187: 627 (1992) all of which arehereby incorporated by reference)

the transmembrane glycoprotein (G) of vesicular stomatitis virus

(Balch et al., J. Biol. Chem., 261: 14681 (1986); Kreis et al., Cell,46: 929 (1986); Doms et al., J. Cell Biol., 105: 1957 (1987); Zhang etal., J. Virol., 68: 2186 (1994); Ohnishi, Curr. Topics Membr. Transp.,32: 257 (1988); Li et al., J. Virol., 67: 4070 (1993); Zagouras et al.,J. Virol., 65: 1976 (1991); Herrmann et al., Biochem., 29: 4054 (1990)all of which are hereby incorporated by reference)

the fusion protein of HIV virus, in particular the gp41 component

(Stareich et al., Cell, 45: 637 (1986); Kowalski et al., Science, 237:1351 (1987); Gallaker et al., Cell, 50: 327 (1987) all of which arehereby incorporated by reference)

the fusion protein of Sendai virus, in particular the F1 component

(Blumberg et al., J. Gene Virol., 66: 317 (1985); Sechoy et al., J.Biol. Chem., 262: 11519 (1987); Homma and Ohuchi, J. Virol., 12: 1457(1973); Scheid and Choppin, Virol., 57: 470 (1974) all of which arehereby incorporated by reference)

the transmembrane glycoprotein of Semliki Forest virus, in particularthe E1 component

(Omar et al., Virol., 166: 17 (1988); Nieva et al., EMBO-J., 13: 2707(1994); Phalen et al., J. Cell Biol., 112: 615 (1991); Lobigs et al., J.Virol., 64: 1233+5214 (1990);, Kenney et al., Structure, 2: 823 (1994);Garoff et al., Nature, 288: 236 (1980); Levy-Mintz et al., J. Virol.,65: 4292 (1991) all of which are incorporated herein by reference)

the transmembrane glycoprotein of tickborn encephalitis virus

(Guirakhoo et al., Virol., 169: 90 (1989); J. Gen. Virol., 72: 1323(1991); Heinz et al., Virol., 198: 109 (1994) all of which areincorporated herein by reference)

the fusion protein of human respiratory syncytial virus (RSV), inparticular the gp37 component

(Collins et al., PNAS, 81: 7683 (1984); Elango et al., Nucl. Acids Res.,13: 1559 (1985) both of which are incorporated herein by reference)

Preparation of Viral-derived Fusion Proteins (c)

Viral-derived fusion proteins are isolated either by dissolving the coatproteins from an enriched viral preparation with the aid of detergents(such as β-D-octylglucopyranoside) and separating them by centrifugation(see, e.g., review in Mannino et al., Bio/Techniques, 6: 682 (1988)hereby incorporated by reference) or else using molecular biologicalmethods which are known to the skilled person. Examples of thepreparation of fusion proteins have already been described for:

influenza hemagglutinin

(Bullough et al., J. Mol. Biol., 236: 1262 (1994); Nature, 371: 37(1994); Daniels et al., Cell, 40: 431 (1985); Godley et al., Cell, 68:635 (1992); White et al., Nature, 300: 658 (1982); Wiley et al., Ann.Rev. Biochem., 56: 365 (1987); Kawaoka et al., PNAS, 85: 321 (1988);Kuroda et al., J. Virol., 63: 1677 (1989), EMBO-J., 5: 1359 (1986);Naeve et al., Virol., 129: 298 (1983); Porter et al., Nature, 282: 471(1972); Hughson, Curr. Biol., 5: 265 (1995) all of which areincorporated herein by reference)

the M2 protein of influenza V

(Black et al., J. Gen. Virol., 74: 1673 (1993); Pinto et al., Cell, 69:517 (1992); Zebedee et al., J. Virol., 56: 502 (1985) all of which areincorporated herein by reference)

the HEF protein of influenza C

(Pfeifer et al., Virus. Res., 1: 281 (1984); Herrler et al., Virol.,113: 439 (1981) both of which are incorporated herein by reference)

the transmembrane glycoprotein of filoviruses, such as

Marburg virus

(Will et al., J. Virol., 67: 1203 (1993); Feldmann et al., Virus Res.,24: 1 (1992) both of which are incorporated herein by reference)

Ebola virus

(Volchkow et al., FEBS Lett., 305: 181 (1992); Virol., 214: 421 (1995);Sanchez et al., Virus Res., 29: 215 (1993); Virol., 157: 414 (1987);Eliott et al., Virol., 163: 169 (1985) all of which are incorporatedherein by reference)

the transmembrane glycoprotein of rabies virus

(Gaudin et al., J. Virol., 65: 4853 (1991); Virol., 187: 627 (1992);Rose et al., J. Virol., 43: 361 (1982); Witte et al., Virol., 185: 681(1991) all of which are incorporated herein by reference)

the transmembrane glycoprotein of vesicular stomatitis virus

(Li et al., J. Virol., 67: 4070 (1993); Riedel et al., EMBO-J., 3: 1477(1984); Lyles et al., Biochem. 29: 2442 (1990); Metsikko et al.,EMBO-J., 5: 3429 (1986) all of which are incorporated herein byreference)

the transmembrane glycoprotein of Semliki Forest virus

(Garoff et al., Nature, 288: 236 (1980); Kielian et al. , J. Virol., 64:4614 (1990); Kondor-Koch, J. Cell Biol., 97: 644 (1983) all of which areincorporated herein by reference)

the transmembrane glycoprotein of tickborn encephalitis virus

(Guirakkoo et al., J. Gen. Virol., 72: 1323 (1991); Heinz et al.,Virol., 198: 109 (1994) both of which are incorporated herein byreference).

Molecules possessing fusiogenic properties are furthermore:

peptides which contain the translocation domain (domain II) ofPseudomonas exotoxin A (Weis et al., Cancer Res., 52: 6310 (1992));Fominaga et al., J. Biol. Chem., 271: 10560 (1996) both of which areincorporated herein by reference)

peptides which contain the peptide (SEQ ID NO:5)

GLFEALLELLESLWELLLEA

(Gottschalk et al., Gene Ther., 3: 448 (1996) hereby incorporated byreference)

peptides which contain the peptide (SEQ ID NO:6)

AALAEA[LAEA]₄LAAAAGC (Acm)

(Wang et al., Technol. Advances in Vector Syst. for Gene Ther., May 6-7,1996, Coronado, IBC Conference, hereby incorporated by reference)

peptides which contain the peptide (SEQ ID NO:7)

FAGV-VLAGAALGVAAAAQI

of the fusion protein of measles virus

(Yeagle et al. , Biochem. Biophys. Acta 1065, 49: (1991) herebyincorporated by reference)

peptides which contain the peptide (SEQ ID NO:8)

GLFGAIAGFIEGGWWGMIDG

of the HA2 protein of influenza A (Lüneburg et al., J. Biol. Chem. 270:27606 (1995) hereby incorporated by reference)

peptides which contain the peptide (SEQ ID NO:9)

GLFGAIAGFIENGWEGMIDGGLFGAIAGFIENGWEGMIDG

(Burger et al., Biochem., 30: 11173 (1991) hereby incorporated byreference) or the peptide(SEQ ID NOS:10-21)

GLFGAIAGFIE;

ALFGAIAGFIE;

LFLGAIAGFIE;

LLLGAIAGFIE;

LILGAIAGFIE;

GIFGAIAGFIE;

GLLGAIAGFIE;

GLFAAIAGFIE;

GLFEAIAGFIE;

GLFGAMAGFIE;

GLFGAIAGLIE or the peptide

GLFGAIAGFIV

(Steinhauer et al., J. Virol, 69: 6643 (1995) hereby incorporated byreference)

or the peptide (SEQ ID NO:22)

GLFEAIAEFIEGGWEGLIEG

or the peptide (SEQ ID NO:23)

GLLEALAELLEGGWEGLLEG

(Ishiguro et al., Biochem., 32: 9792 (1993) hereby incorporated byreference).

Conjugation of the Ligands and Fusion Proteins to the Carrier

The ligands and fusion proteins are conjugated to the carrier usingmethods that are known to the skilled person.

Examples of Non-covalent Bonds

carrier-biotin←→ avidin-S-S-ligand Hashimoto et al., 132: 129 (1984),hereby incorporated by reference carrier←→ bispec. Raso et al., Immunol.antibody ←→ligand Rev., 62: 93 (1982) hereby incorporated by referenceExamples of covalent bonds bonding to protein NH₂ groups Carlson et al.,Biochem. J., 173: 723 (1978) hereby incorporated by reference necessaryreagent: N-succinimidyl-3-(2-pyridylthio)propionate (SPDP)SDDP-dithiothreitol Carlson et al., above 2-iminothiolane King et al.,Biochem., 17: 1499 (1978) hereby incorporated by reference2,2-iminothiolane + 4,4 dithiopyridine King et al., Biochem. 17: 1499(1978) hereby incorporated by reference 3-methyl-3-(4-dithiopyridyl)-mercaptopropionimidate N-acetylhomocysteine thiolactone Reiner et al.,J. Mol. Catal., 2: 335 (1977) hereby incorporated referenceacetylmercaptosuccinic Klotz et al., Arch. Biochem. anhydride + NH₂OHBiophys., 96: 690 (1979) hereby incorporated by referencem-maleimidobenzoyl-N- Liu et al., Biochem. hydroxysuccinimide ester 18:690 (1979) hereby incorporated by reference succinimidyl-4-(N-maleimido-Yoshitake et al., Eur. methylcyclohexane)-1-carboxylate J. Biochem.,101: 395 (1979) hereby incorporated by referenceN-succinimidyliodoacetate Rector et al., J. Immun. Meth., 24: 321 (1978)hereby incorporated by reference 4-hydroxy-3-nitromethylbenzimidate +Müller et al., acetimidate + Na₂S₂O₄ J. Appl. Biochem., 1: 301 (1979)hereby incorporated by reference 4-hydroxy-3-nitromethylbenzimidate +Müller et al. above acetimidate + NaNO₂ oxidized dextran + borohydrideHurwith et al., Eur. J. Cancer, 14: 1213 (1978) hereby incorporated byreference Bonding to protein hydroxyl groups necessary reagent:cystamine + carbodiimide Erlanger et al., Meth. Imm. Immunochem., 1: 144(1967) hereby incorporated by reference Gilliland, Cancer Res., 40: 3564(1980) hereby incorporated by reference Bonding to protein SH groupsnecessary reagent: Protein SH Ghose and Blair, CRC Crit. Rev. Ther. DrugCarrier Syst., 3: 263 (1987) hereby incorporated by reference ProteinSH + Na₂S₄O₆ Masuko et al., BBRC, 90: 320 (1979) hereby incorporated byreference Ellman's reagent Raso and Griffin, J. Immunol., 125: 2610(1980) hereby incorporated by reference Bonding to protein aldehydegroups necessary reagent: periodate Hurwitz et al., Cancer Res., 35:1175 (1975) hereby incorporated by reference Bonding to protein COOHgroups necessary reagent: cystamine + carbodiimide Gilliland, CancerRes., 40: 3564 (1980) hereby incorporated by reference

Choice of the Nucleotide Sequences for the Gene (d) which is to beIntroduced

The nucleotide sequences that are to be complexed with the carrier canbe DNA sequences or RNA sequences. In the simplest case, they comprisenaked nucleotide strands that contain the gene that encodes the desiredprotein. This gene can be supplemented with cell-specific orvirus-specific promoter sequences and, furthermore, with promotermodules.

Furthermore, viral promoter sequences and/or enhancer sequences can beadded to the gene in order to amplify and/or extend expression of thegene. Promoter sequences and/or enhancer sequences of this nature arereviewed, for example, by Dillon, TiBTech, 11: 167 (1993) herebyincorporated by reference. Examples of promoter sequences and/orenhancer sequences of this nature are:

the LTR sequences of Rous sarcoma viruses

the LTR sequences of retroviruses

the promoter region and enhancer region of CMV viruses

the ITR sequences and/or the p5, p19 and p40 promoter sequences of AAVviruses

the ITR sequences and/or promoter sequences of adenoviruses

the ITR sequences and/or promoter sequences of vaccinia viruses

the ITR sequences and/or promoter sequences of herpesviruses

the promoter sequences of parvoviruses

the promoter sequences (upstream regulator region) of papilloma viruses

Preferably, the gene is incorporated into a plasmid.

Complexing the Conjugated Carriers (a) with the Gene (d)

The conjugated carrier is complexed with the gene, or the nucleotidesequence, by mixing the two starting substances. A mixing ratio shouldpreferably be chosen which results in complexes which have a neutral orcationic charge.

Examples of preferred mixing ratios are:

1 μmol of lipid/20 μg of plasmid

1-5 mg of lipid/10-20 μg of DNA/RNA

6.2 μg of lipid/1.55-3.1 μg of DNA

lipid/DNA-peptide (5:1)

The loading is effected by incubating the positively charged carrierwith genes in the desired mixing ratio. The mixing ratio is determined(as described by Dittgen et al., Pharmazie, 42: 541 (1987) herebyincorporated by reference, by zeta potential measurement.

The following examples are intended to exemplify particular embodimentsof the invention which is defined by the specification and appendedclaims.

EXAMPLE 1 Preparation of an Active Compound for Transfecting EndothelialCells

a) Preparation of the Filovirus Glycoprotein as the Ligand

The filovirus glycoprotein is a coat protein which has a high affinityfor endothelial cells. The filovirus glycoprotein is prepared asdescribed in detail by Will et al., J. Virol., 67: 1203 (1993); Feldmannet al., Virus Res., 24: 1 (1992) and Volchkow et al., FEBS Lett., 305:181 (1992) all of which are hereby incorporated by reference.

b) Preparation of Ebola Viruses

The Ebola virus subtype “Zaire” (EBO, Institute for Virology, SergievPosad, Russia) was passaged in macaque rhesus monkeys and then culturedin Verocells and isolated from the cell culture liquid (Volchkow et al.,FEBS Lett., 305: 181 (1992) hereby incorporated by reference).

c) Cloning and Sequencing the Viral RNA

Genomic RNA was isolated from purified viruses by centrifugation throughcesium chloride gradients (Volchkow et al. (1992) incorporated byreference). This RNA was employed to prepare a CDNA library using randomprimers and a chick myeloblastosis virus reverse transcriptase. RNA-cDNAhybrids from the cDNA library were used as starting material foramplifying the GP gene with the aid of PCR and the following syntheticprimers.

N1, having the sequence

5′-GAAGGATCCTGTGGGGCAACAACACAATG (Seq.ID-No. 1)

(complementary to nucleotides 114 to 142 of the mRNA sense) supplementedwith a 5′-terminal BamH1 region.

N2, having the sequence

5′-AAAAAGCTTCTTTCCCTTGTCACTAAA (Seq.ID-No. 2)

(complementary to nucleotides 2492 to 2466 of the mRNA sense)supplemented with a 5′-terminal Hind-III region.

The DNA nucleotide sequence of the GP gene was analyzed, for bothstrands, using the Maxam and Gilbert method (Methods in Enzymology, 65:499 (1980) hereby incorporated by reference). The sequence of the EBOZaire strain GP gene was deposited in the gene bank under no. U31033(Volchkow et al. (1992) hereby incorporated by reference).

The sequence of the EBO GP gene was to a large extent identical to thatpublished by Sanchez et al. (Virus Res. 29, 215 (1993) herebyincorporated by reference). However, only seven, rather than eight,consecutive A's (adenine; MRNA sense) were found in positions 1019 to1025.

d) Isolation, Cloning and Sequencing of the mRNA which is Specific forthe EBO-GP

Using the RNeasy total RNA kit (from Quiagen), the mRNA for the EBO-GPwas isolated from about 7×107 Verocells which were infected with the EBOvirus (1-10 PFU per cell, 1 day post-infection).

For the CDNA synthesis 10 μl of the mRNA solution (corresponding toabout 1.4×10⁷ infected cells) were incubated with chick myeloblastosisvirus reverse transcriptase in the presence of the primer

N3, having the sequence

oligo-d (T)21, supplemented with a 5′-terminal Hind-III region.

The RNA was subsequently removed by incubating the mixture with 1 μg/μlRNAse at 37° C. for 30 min.

The GP-specific nucleotide sequence was amplified by means of PCR usingprimers N1 and N3.

The PCR was carried out in a 100 μl reaction mixture containing 1-5 μgof cDNA in 50 mM KCl; 10 mM Tris-HCl (pH 8.3), 2 mM MgCl₂; 0.2 mM ofeach deoxynucleotide and 0.3 μM of each primer.

The reaction mixture was heated at 95° C. for 10 min. and Taq polymerase(2.5 U/100 μl) was added. 35 cycles of DNA amplification were carriedout. The cycle program comprised 94° C. for 1 min.; 70° C. for 1 min.and 72° C. for 1 min. After the cycle program, the samples wereincubated at 72° C. for 10 min. (All the components of the PCR wereobtained from Perkin-Elmer Cetus). The products of the PCR reaction werepurified (QIA Quick Spin PCR purification kit; from Quiagen) and the DNAwas used directly for the sequencing, for repeat PCR reactions or forcloning plasmid vectors. The nucleotide sequence of the GP region, whichoverlapped in ORF's (“open reading frames”) I and II, was determinedusing the Sanger technique (Sanger et al., PNAS 74, 5463 (1977) herebyincorporated by reference), with the following primers being employed:

N4, having the sequence

5′-CGGACTCTGACCACTGAT (Seq. ID-No. 3)

(complementary to nucleotides 1108 to 1091) N5, having the sequence

5′-TCGTGGCAGAGGGAGTGT (Seq. ID-No. 4)

(complementary to nucleotides 1412 to 1395).

The PCR fragments were cloned into the pGEM2Zf(+) ector, and therecombinant plasmids were analyzed by eans of enzymic sequencing (Sangeret al., above.

Most of the clones exhibited (just like the vRNA) 7 consecutiveadenosines between positions 1018 and 1026 (MRNA sense) whereas 8consecutive adenosines were found in this position in about 20% of thecells which were infected with GP-specific EBO mRNA.

The mRNA containing 8 adenosines encodes a complete GP of 676aa (sincethe 8th adenosine enables the frameshift from ORF I to ORF II to takeplace).

By contrast, the mRNA containing only 7 adenosines encodes anon-structural, second glycoprotein (sGP). In conformity with thenucleotide sequence of sGP, the sGP appears to be identical to theN-terminal part (274aa) of GP, supplemented by an additional 70aa whichare encoded by the end of the ORF I.

This end is not present in the GP, since, in this latter case, theadditional 8th adenosine transfers reading from ORFI to ORF II, and ORFI is consequently not read to the end.

e) Construction of Recombinant Plasmids

The PCR products, which constitute the complete ORF of the EBO GP gene,were incubated with restriction enzymes Bam H I and Hind III and ligatedinto the plasmid pGEM3Zf(+), which had been pretreated with Bam H I andHind III. The plasmid contains a T7 phage RNA polymerase promoter, whichwas employed to synthesize the EBO GP-RNA with T7 polymerase using thevaccinia virus/T7 polymerase expression system.

Plasmids which contained the complete GP nucleotide sequence of the EBOvRNA (7 consecutive adenosines) were designated pGEM-mGP7, and thosewhich correspondingly contained the EBO mRNA (8 consecutive adenosines)were designated pGEM-mGP8.

The GP-specific nucleotide sequences were excised from plasmidspGEM-mGP7 and pGEM-MGP8 using Bam H 1 and Hind III and the ends of theresulting fragments were filled using the Klenow fragment of DNApolymerase I; the sequences were then linked to the Smal restrictionsite of vector pSc 11 (from Promega, Madison, Wis.). The resultingrecombinant plasmids (pSc-mGP7 and pSc-mGP8) were, used for preparingrecombinant vaccinia viruses.

f) Construction of Recombinant Vaccinia Viruses

Recombinant vaccinia viruses were prepared by means of homologousrecombination between the tK regions in the recombinant plasmids(pSC-mGP7 or pSC-mGP8) and the genomic DNA of vaccinia virus (WR strain,as described by Chakrabarti et al. (Mol. Cell. Biol., 5: 3403 (1985)hereby incorporated by reference) and using the lipofectin transfectionmethod (Felgner et al., PNAS, 84: 7413 (1987)).

Recombinant viruses were purified by passaging once on TK-143 cells andpassaging four times on CV-1 cells using β-galactosidase-positiveplaques for the selection.

Recombinant vaccinia viruses which derive from plasmid pSC-mGP7 weretermed vSC-GP7, while those which derive from plasmid pSC-mGP8 weretermed vSC-GP8.

Expression of GP was achieved by infecting 1×10⁶ Hela cells, or the samenumber of RK-13 cells, with 10 pfu of vSC-GP7 or vSC-GP8 per cell.

The expression of the gene products was analyzed by means of theimmunoblot method. For this, lysates of 1.4×10⁵ infected Hela cells orRK-13 cells were fractionated on 10% SDS-PAGE (as described by Laemmli,Nature, 227: 680 (1970) hereby incorporated by reference) and loadedonto PVDF membranes (from Millipore) in accordance with the semidrytechnique. Secreted sGP was analyzed by loading 20 μl of the supernatant(2 ml) from 1×10⁶ infected cells onto the gel. The immunoanalysis wascarried out using a mouse anti-EBO serum or a horse anti-EBO serum and arabbit anti-mouse or anti-horse antibody as the second antibody, in eachcase conjugated with horseradish peroxidase. The bound second antibodywas analyzed using the ECL technique (from Amersham).

It was possible to detect both GP and sGP in cell lysates of both theinfected Hela cells and the infected RK-13 cells, with the proportion ofsGP being markedly greater after infection with vSG-GP7 and that of GPbeing markedly greater after infection with vSC-GP8.

Endoglycosidase H treatment of the cell lysates and SDS-PAGE analysisindicated that the mature GP has a molecular weight of 125-140 kD and(in contrast to “immature” GP) is resistant to cleavage withendoglycosidase H owing to the complex, N-glycosidically bondedoligosaccharides.

RK-13 cells express a mature GP having a molecular weight of 140 kD,whereas Hela cells express a mature GP of 125 kD. The differences insize are due to N-glycosylation which differs in a cell-specific manner.The 140 kD GP from RK-13 cells comigrated in SDS-PAGE with the GP ofEbola viruses.

Cells which were infected vSC-GP7 exhibited only small quantities of sGPin the cell lysate. This sGP always had a molecular weight of 50 kD. Theoverwhelming proportion of the sGP was to be found in the cellsupernatant (the ratio of secreted sGP to intracellular sGP was 25:1).Secreted sGP has a molecular weight in the range of 50-55 kD and isresistant to endoglycosidase H.

g) Selection and Purification of the GP

EBO-GP, MW 140 kD, produced by RK-13 cells, was selected as the ligandfor preparing the novel non-viral vector.

RK-13 cells were infected with 10 pfu of the vSC-GP8/cell. The cellswere harvested and lysed at from 16 to 18 hours after infection. Theproteins in the lysate were fractionated on a preparative 8% SDS-PAGEand stained with Coomassie brilliant blue. The GP was excised and thegel pieces were placed in a BioTrap (from Schleicher and Schüll); thelatter was in turn placed in a horizontal electrophoresis chamber. Theelectroelution was carried out in a buffer (100 mM glycine, 20 mM TRISand 0.01% SDS), at 4° C. and constant voltage (200 V), for from 16 to 20hours. The eluate was collected and concentrated using a Centricon-100microconcentrator (from Amicon).

A sample of the concentrated eluate was subjected to electrophoreticfractionation on 10% SDS-PAGE and examined for purity by means ofstaining with Coomassie brilliant blue and immunoblotting.

Thereafter, the GP was ultrapurified by means of reversed phase HPLC [WP300 column (0.46×25 cm), C4.5 μm (from Shandon)] using an acetonitrilegradient (from 0% to 100% B in 40 min.; A:0.1% TFA, 10% acetonitrile;B:0.1% TFA; 90% acetonitrile) at 60° C. and with a flow-through rate of1 ml/min.

EXAMPLE 2 Preparation of the Fusion Protein M2 of Influenza A

The M2 protein is prepared as described in detail by Zebedee et al., J.Virol., 56: 502 (1985); Pinto et al., Cell, 69: 517 (1992) and Black etal., J. Gen. Virol., 74: 1673 (1993), all of which are herebyincorporated by reference.

EXAMPLE 3 Preparation of a Protein (albumin)-based Cationized Carrier

The carrier system is prepared and characterized as described by Muller,Dissertation, University of Basle (1994). The particles werecharacterized with regard to their size (photon correlationspectroscopy), surface charge (zeta potential measurement) andmorphology (scanning electromicroscopy) in accordance with the methodsknown to the skilled person, as described in Müller (1994) above andJunginger et al., Pharm. Ztg., 25: 9 (1991) both of which are herebyincorporated by reference.

Albumin was cationized as described in detail by Bergmann et al., Clin.Sci., 67: 35 (1984) and Kumagai et al., J. Biol. Chem., 262: 15214(1987) both of which are hereby incorporated by reference. Afteractivating the carboxyl groups with N-ethyl-N′-3-(dimethylaminopropyl)carbodiimide hydrochloride, human serum albumin was positivized by thecovalent coupling of hexamethylenediamine. Unreacted constituents wereremoved by means of dialysis and column chromatography. The extent ofthe cationization can be ascertained by determining the zeta potentialand by means of electrophoretic methods.

3.1. Preparation of Cationized Human Serum Albumin (cHSA)

Human serum albumin (5 ml of a 20% solution) is added to 67 ml of a 2 Msolution of hexamethylenediamine and the pH is adjusted to 7.8. Themixture is stirred at room temperature and 100 rpm for 30 min andtreated with 1 g of N-ethyl-N′-3-(dimethylaminopropyl)carbodiimidehydrochloride. After checking the pH once again, the mixture is stirredat room temperature for 4 h, with overheating being avoided by means ofoccasional incubations in an ice bath. The end product is concentrated,and purified from low molecular weight constituents, in an Amiconconcentrator (MWCO 30.000) in five centrifugation steps. Thepurification procedure is monitored photometrically (280 nm) andosmometrically. The purified derivative is lyophilized and stored at 4°C.

3.2. Characterization of cHSA

The cationized albumin is characterized by means of electrophoresistechniques which are known from the literature. Isoelectric focusingusing homogeneous polyacrylamide gels (5% T, 3% C) and PharmalyteCarrier Ampholytes in a pI range of 3-9 (Phast Gel IEF 3-9) shows thatthe isoelectric point has been displaced from pl 4 to pl 7-9. Themolecular weight of the albumin of 67.000, as determined by SDS-PAGE(Phast Gel gradient 10-15, Phast Gel SDS buffer strips, non-reducing),is not altered by the cationization. Higher molecular weight aggregatesare not detectable using the same technique.

The UV absorption maximum of the cationized albumin is 276-278 nm. Theextent of the cationization is quantified fluorimetrically by reactingthe primary amine functions with fluorescamine at 390 nm(excitation)/475 nm (emission) and at pH 7. A conversion factor of2.0-2.3 suggests that reaction has been complete.

The BCA protein assay, which is known from the literature, can be usedto determine cationized albumin, for example in a mixture with plasmids,photometrically at 562 nm, without interference, in a concentrationrange of 5-10 μg/ml.

3.3. Checking the Function of the cHSA

a) Preparation of Macromolecular plasmid/cHSA Complexes

CMV-lacZ plasmid (22.95 μg) is incubated, at room temperature and at 100rpm, for 10 min in 1.4 ml of sterile 0.9% sodium chloride solution. 1 mlof a solution, which has been sterilized by filtration, of thecationized human serum albumin in 0.9% sodium chloride solution is addedto the plasmid solution at a drop rate of one drop per second, and themixture is complexed at room temperature for 5 min while being stirred.

b) Characterization of the Complexes

The concentration of cHSA which is required for neutralizing andcationizing the plasmid is determined by means of agarose gelelectrophoresis (1% agarose gel, TAE buffer, pH 7.4, 90 V, 3 h). Unboundplasmid constituents are detected by ethidium bromide intercalationwhile protein constituents are detected by a subsequent Coomassiestaining. Under the chosen conditions, plasmid/protein complexes havinga positive overall charge migrate to the cathode in a clearly visiblemanner. While complexes which are prepared using 0.3 ml of the 8.3 mg/mlcHSA solution exhibit a negative net charge, complexes which areprepared with 0.6 ml, 1 ml, 1.5 ml and 2 ml of the solution exhibit apositive net charge. Free plasmid constituents do not appear at any ofthe concentrations selected.

The plasmids are not damaged in a detectable manner by being incubatedfor 10 minutes in physiological sodium chloride solution.

The complex size which is suitable for transfection is determined bymeans of photon correlation spectroscopy (Zeta sizer 1, AZ 110, 90°,wavelength 633). The complex size is adjusted to 310-360 nm by varying

the incubation volume

the nature and ionic strength of the incubation medium

the cHSA concentration

the period of incubation with cHSA

the rate at which the cHSA solution is injected

and the pH of the incubation medium.

Opalescent solutions are produced.

c) Transfection of Cells with cHSA/CMV-lacZ Complexes

3T3 cells are cultured in DMEM containing 10% FCS pH 7.1, withoutantibiotics being added, at 37° C., 5% CO₂ and 89% humidity.

d) Gene Transfer

3T3 cells 200,000 were sown in gelatin-coated 3 cm Petri dishes andcultured for 24 h until approx. 50% confluence is reached. The mediumwas removed and the cells washed 3 times with PBS without calcium andmagnesium, pH 7.4, and then treated with 2 ml of DMEM without FCS orwith 1 ml of 150 mM NaCl/lo mM HEPES, pH 7.4. In each case, the mixturewas diluted with 0.84 ml of the plasmid/cHSA complex described under2.1, corresponding to 8 μg of DNA, and incubated at 37° C. for 1 h(NaCl/HEPES) or 2 h (DMEM).

In addition, various agents such as 85% glycerol (sterilized, 200 μl,1.84 ml) were added to the transfection mixture in order to modifyfusiogenic and lysosomotropic properties. After the incubation, thetransfection medium was sucked off and replaced with DMEM containingFCS, after which the whole was incubated for a further 48 h. In order tosimulate lysosomotropic effects, a portion of the mixtures wasincubated, after the transfection period of 1 or 2 hours, for a further3 h with chloroquine-containing DMEM (DMEM, 2.5% FCS, 0.1 mMchloroquine). After that, the chloroquine solution was replaced withDMEM containing 10% FCS.

In order to check the experimental conditions, the 3T3 cells weretransfected using lipofectamine in accordance with a method known fromthe literature (10 μl of reagent, 2 μg of DNA).

After 48 h, the cell culture medium was removed and the cells washed 1×with PBS without calcium and magnesium, pH 7.4, and fixed for 10 minwith 0.1% glutaraldehyde in PBS. Excess glutaraldehyde was removed by afurther, double wash with PBS.

Transfected cells were stained by incubating them with a solution of 3.0mM potassium ferricyanide, 3.0 mM potassium ferrocyanide and 0.08% X-Gal(stock: 2% in DMF) in PBS at room temperature overnight and are thenassessed microscopically.

In no case did pure plasmid/cHSA complexes result in transfection. Theaddition of 85% glycerol to the cell culture medium resulted in thedeath of the cells after only 1 h. Transfection rates corresponding tothe values in the literature which were obtained using DEAE dextran canbe observed in NaCl/HEPES-buffered solution after chloroquine shock.Using the same aftertreatment, gene transfer is considerably lesspronounced in DMEM.

TABLE 1 Overview of the characterization of the cationized human serumalbumin Method Result Solubility — good solubility in water andphysiological media p1 isoelectric p1 7-9 focusing Molecular weightSDS-PAGE 67,000 Higher molecular SDS-PAGE no aggregates weightaggregates detectable Absorption maximum UV spectroscopy 276-278 nmExtent of the fluorimetry theoret.: 2.18 cationization (fluorescamine)pract.: 2.0-2.3 Determination of agarose gel positive charge the totalcharge electrophoresis Content BCA protein assay — determination

Table 2: Validation of the conditions for the complex formation

Incubation volume:

110 μl

1.4 ml

Nature and ionic strength of the incubation medium:

double-distilled water

PBS without calcium and magnesium, pH 7.4

physiological saline solution

150 mM NaCl, 10 mM HEPES, pH 7.4

DMEM without serum, pH 7.4

cHSA concentration:

Concentrations tested:

0.1 μg, 1 μg, 10 μg, 100 μg, 0.5 g, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 5mg and 10 mg, in each case per ml.

Concentrations selected:

2.49 mg, 5.16 mg, 8.3 mg, 12.45 mg and 16.6 mg, in each case per ml.

Incubation period

5, 30, 60, 120 and 180 min

pH of the incubation medium:

pH 4.0

pH 7.4

pH 9

Injection rate

1 drop/sec

complete addition of CHSA immediately

EXAMPLE 4 Preparation of the Plasmid

The human endothelin-1 promoter (position <−170 to >−10; Wilson et al.,Mol. Cell Biol., 10: 4654 (1990), hereby incorporated by reference,) ora variant which has been truncated by the length of the TATA box(position <−170 to >−40) is linked, at its 3′ end, to the 5′ terminus ofthe CDE-CHR-Inr module (position <−20 to >−+121) of the human cdc25Cgene (UK 950.6466.3). The linkage is effected using enzymes which areknown to the skilled person and are commercially available.

The chimeric endothelin-1 promoter module/transcription unit which wasprepared in this way was linked, at its 3′ end, to the 5′ terminus of aDNA which contained the complete coding region of human β-glucuronidase(position <27 to >1982; Oshima et al., PNAS USA, 84: 685 (1987) herebyincorporated by reference). This DNA also contains the signal sequence(22 N-terminal amino acids) which is required for secretion. In order tofacilitate secretion from the cell, this signal sequence was exchangedfor the immunoglobulin signal sequence (position <63 to >107; Riechmannet al., Nature 332, 323 (1988) hereby incorporated by reference).Transcription control units and the DNA for β-glucuronidase were clonedinto pUC18/19 or Bluescript-derived plasmid vectors using enzymes whichare known to the skilled person and are commercially available.

EXAMPLE 5 Complexing the Cationized Carrier with the Plasmid

The carrier is complexed with the plasmid by incubating the twocomponents in a suitable mixing ratio. The extent of the association wasascertained from the alteration in the zeta potential.

The cationic HSA (6.5 ml of a 20% solution prepared as described underc) and selected from a 5-35% range) were treated with the plasmidsolution in a ratio of 1:2 (selected from a range of from 1:1 to 1:10).The outer phase, consisting of 93.5 ml of dichloromethane/methanol (9:1)containing 0.5% Klucel GF, was temperature-equilibrated at 20° C. for 30min and the albumin solution was added to the organic phase, which wascirculating with a throughput of 500 ml/min. The emulsion was sonicatedin a pulsed manner at 65 watt for 15 min. For the crosslinking, 6.6 mmolof glutaraldehyde in methylene chloride were added to the mixture andthe whole was stirred at 2200 rpm, at room temperature, for 80-100 min.The particles were purified by being washed and centrifuged down severaltimes.

EXAMPLE 6 Introduction of Lipophilic Groups

Lipophilic groups were introduced by acylating under conditions known tothe skilled person. In this context, the carboxylic acid derivativereacts with the primary amino groups of the albumin in accordance withthe known addition-elimination mechanism of acylation to form thecarboxamide.

Oleoyl chloride 0.1 g was dissolved in 5-10 ml of anhydrous dioxane andthis solution was treated dropwise, in a ratio of 1:4 (selected from arange of 1:1-1:10), with a suspension of the particles (prepared asdescribed under c)) in dioxane; the mixture was then shaken vigorously.After an excess of an aqueous solution of ammonia had been added, themixture was stirred for 10 min and slightly acidified with dilutehydrochloric acid. The particles were separated off by centrifugationand washed with water until neutral.

EXAMPLE 7 Conjugation of Ligands and Fusion Protein to the Carrier

Ligands and fusion proteins are linked to the carrier system by covalentcoupling in accordance with the SPDP method, as described in Khawli etal., Int. J. Rad. Appl. Instrum. B, 19: 289 (1992), and Candiani et al.,Cancer Res., 62: 623 (1992) both of which are hereby incorporated byreference. In this context, primary amino functions present in thelysine residues of the albumin react with SPDP to formdisulfide-containing derivatives. These latter can be bonded covalentlyto sulfhydryl groups of the ligands and fusion proteins under conditionswhich are known to the skilled person.

SPDP reagent (200 nmol in 99.5% ethanol) was incubated, at roomtemperature for 30 min, with 74 nmol of cationized, lipophilized HSAparticles (prepared as described under f)) in PBS. For the conjugation,the mixture was treated with a solution of the Ebola virus GPglycoprotein (prepared as described under a)) and the M2 fusion proteinof influenza (prepared as described under b)) in a ratio of 1:1(selected from a range of 1:1-1:10) and the whole was incubated at roomtemperature overnight and while being stirred. Unbound constituents werecentrifuged off.

EXAMPLE 8 Activity of the Target cell-specific Vector

The target cell-specific vector, prepared as described in the precedingexamples, preferentially binds, following systemic, preferablyintravenous or intraarterial administration, to endothelial cells bymeans of a tissue-specific ligand (b). Following uptake into theendosomes, penetration into the cytoplasm takes place which is mediatedby the fusion protein (c). The tissue-specific promoter sequence, andthe cell cycle-regulated promoter module, ensure that the gene (d) ismainly expressed in proliferating endothelial cells. The presence ofthese genes results in these proliferating endothelial cells secretingβ-glucuronidase, which cleaves pharmacologically inactiveβ-glucuronidides (prodrugs) into active substances. This activesubstance can, for example, have an antiproliferative or cytostaticeffect. This results in inhibition of proliferation of the endothelialcell and inhibition of the growth of a neighboring tumor or inhibitionof an adjacent inflammatory reaction. Since the novel active compoundrestricts production of the antiproliferative or cytostatic substance tothe site of the angiogenesis which is caused by the tumor or theinflammation, it is well tolerated.

Choosing the (non-viral) carrier for the selected gene results in therebeing no risk of the patient's genes being mutated due to activation ofquiescent viruses which are integrated in the genome or due torecombination with wild-type viruses.

Priority document, Federal Republic of Germany application No.19605279.3, filed Feb. 13, 1996, including the specification, claims,abstract & drawings, is hereby incorporated by reference.

23 29 base pairs nucleic acid single linear 1 GAAGGATCCT GTGGGGCAACAACACAATG 29 27 base pairs nucleic acid single linear 2 AAAAAGCTTCTTTCCCTTGT CACTAAA 27 18 base pairs nucleic acid single linear 3CGGACTCTGA CCACTGAT 18 18 base pairs nucleic acid single linear 4TCGTGGCAGA GGGAGTGT 18 20 amino acids amino acid single linear 5 Gly LeuPhe Glu Ala Leu Leu Glu Leu Leu Glu Ser Leu Trp Glu Leu 1 5 10 15 LeuLeu Glu Ala 20 29 amino acids amino acid single linear 6 Ala Ala Leu AlaGlu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala 1 5 10 15 Glu Ala LeuAla Glu Ala Leu Ala Ala Ala Ala Gly Cys 20 25 19 amino acids amino acidsingle linear 7 Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val AlaAla Ala 1 5 10 15 Ala Gln Ile 20 amino acids amino acid single linear 8Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Trp Gly 1 5 1015 Met Ile Asp Gly 20 40 amino acids amino acid single linear 9 Gly LeuPhe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 MetIle Asp Gly Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn 20 25 30 GlyTrp Glu Gly Met Ile Asp Gly 35 40 11 amino acids amino acid singlelinear 10 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 aminoacids amino acid single linear 11 Ala Leu Phe Gly Ala Ile Ala Gly PheIle Glu 1 5 10 11 amino acids amino acid single linear 12 Leu Phe LeuGly Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 amino acids amino acid singlelinear 13 Leu Leu Leu Gly Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 aminoacids amino acid single linear 14 Leu Ile Leu Gly Ala Ile Ala Gly PheIle Glu 1 5 10 11 amino acids amino acid single linear 15 Gly Ile PheGly Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 amino acids amino acid singlelinear 16 Gly Leu Leu Gly Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 aminoacids amino acid single linear 17 Gly Leu Phe Ala Ala Ile Ala Gly PheIle Glu 1 5 10 11 amino acids amino acid single linear 18 Gly Leu PheGlu Ala Ile Ala Gly Phe Ile Glu 1 5 10 11 amino acids amino acid singlelinear 19 Gly Leu Phe Gly Ala Met Ala Gly Phe Ile Glu 1 5 10 11 aminoacids amino acid single linear 20 Gly Leu Phe Gly Ala Ile Ala Gly LeuIle Glu 1 5 10 11 amino acids amino acid single linear 21 Gly Leu PheGly Ala Ile Ala Gly Phe Ile Val 1 5 10 20 amino acids amino acid singlelinear 22 Gly Leu Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp GluGly 1 5 10 15 Leu Ile Glu Gly 20 20 amino acids amino acid single linear23 Gly Leu Leu Glu Ala Leu Ala Glu Leu Leu Glu Gly Gly Trp Glu Gly 1 510 15 Leu Leu Glu Gly 20

What is claimed is:
 1. A macrophage or lymphocyte cell-specificnon-viral vector for inserting at least one exogenous gene into amacrophage or lymphocyte, said vector comprising a complex of thefollowing components: a) a non-viral carrier for said exogenous gene, b)a ligand that has a high specific affinity for said macrophage orlymphocyte, wherein the ligand is selected from the group consisting ofIL-2, IL-4, IL-10, M-CSF, and a glycoprotein from the coat of a virusselected from the group of viruses consisting of HIV-2, varicella zostervirus, herpesvirus 6, HTLV-II, HTLV-I, Epstein-Barr virus, and the gp120 protein of HIV-1, c) a fusion protein or a fusiogenic peptide thatpromotes the penetration of said vector into the cytoplasm of saidmacrophage or lymphocyte, and d) said exogenous gene.
 2. The vector asclaimed in claim 1, wherein the individual components of the targetcell-specific vector are bonded to each other covalently, by adsorptionforces, or by ionic interaction.
 3. The vector as claimed in claim 1,wherein said non-viral carrier (a) for said gene is selected from thegroup consisting of a protein, polypeptide, polysaccharide,phospholipid, cationic lipid, glycoprotein, lipoprotein andlipopolyamine.
 4. The vector as claimed in claim 1, wherein saidnon-viral carrier (a) possesses a lipophilic side group that is bondedby adsorptive, ionic or covalent bonding, whereby said carrier is givenamphiphilic properties.
 5. The vector as claimed in claim 1, whereinsaid non-viral carrier (a) is albumin or xylan.
 6. The vector as claimedin claim 1, wherein said fusion protein (c) is selected from the groupconsisting of hemagglutinin of influenza A or B viruses, the HA2component of the hemagglutinin of influenza A or B viruses, a peptideanalogue thereof, the M2 protein of influenza A viruses, the HEF proteinof influenza C viruses, a transmembrane protein of filoviruses, atransmembrane glycoprotein of rabies virus, vesicular stomatitis virus,Semliki Forest virus, tickborn encephalitis virus, a fusion protein ofHIV virus, Sendai virus, respiratory syncytial virus, and a fragment ofsaid viral fusion proteins or of transmembrane glycoprotein from saidviruses from which the transmembrane region is removed.
 7. The vector asclaimed in claim 1, wherein said gene (d) which is to be inserted is inthe form of a plasmid.
 8. A vector according to claim 1, wherein saidmacrophage or lymphocyte is in an organism.
 9. The vector as claimed inclaim 3, wherein said non-viral carrier (a) is given a positive chargeby introducing into said carrier a positively charged side chain, suchthat the bonding between said non-viral carrier and said positivelycharged side chain is effected by adsorptive, ionic or covalent bonding.